This proposal will focus on studies of the structure of human factor VIII (antihemophilic factor) and mechanisms that regulate coagulant activity. Specific aims will assess inter-subunit interactions in factor VIII and in the activated cofactor, factor VIII-a; regulation of factor VIII-a activity by factor IX-a; and mechanisms for cofactor inactivation catalyzed by activated protein C (APC). Studies on the purified subunits of the factor VIII heterodimer will determine equilibrium constants, assess the nature of heavy chain instability and determine whether light chain cleavage by thrombin contributes to the potentiation of activity. Factor VIII-a is a trimer of Al,A2 and A3-ClC2 subunits and the dissociation of A2 subunit from the Al/A3-Cl-C2 dimer accounts for the spontaneous decay of activity. The hypothesis that the acidic terminal region of Al subunit is required for A2 subunit retention will be tested using synthetic peptides to probe this interaction. Comparison of the Me2+-dependent interaction within the factor VIII heterodimer and the Al/A3-Cl-C2 dimer will determine interactive contributions made by the A2 domain. Factor IX-a (transiently) stabilizes factor VIII-a and its effect on the association and dissociation rate constants for the A2-Al/A3-Cl-C2 interaction will be determined. Subsequent inactivation results from cleavage of Arg336 (Al subunit), a site also attacked by APC and the efficiency of the two proteases will assess the physiologic importance of factor IX-a inactivation. Reconstitution assays will determine whether the lagging cleavage of A3 by factor IX-a contributes to cofactor inactivation. Fluorescence energy transfer techniques will be used to examine the topography of the factor Xase complex and determine equilibrium constants for the human factor VIII-a-factor IX-a interaction. Furthermore, a putative factor IX-a/X binding domain within the A2 subunit will be investigated. The inactivation of factor VIII-a by APC may result from destruction of a macromolecular interactive site (cleavage in A2) and loss of the A2 subunit binding site (cleavage in Al) and this hypothesis will be tested following correlation of loss. of these properties with specific bond cleavage. Additional studies will examine APC-catalyzed inactivation of factor VIII-a in the factor Xase complex and assess the potential for (transient) factor IX. protection and the role of protein S in this reaction. Overall, these studies will offer insights into a coagulation protein central to hemostasis.